1. Technical Field
The present invention relates generally to a method, and reagents useful in the method, for determining ligands in liquids, especially biological fluids such as serum, plasma, spinal fluid, amnionic fluid and urine. The present invention relates more particularly to a novel fluorescence polarization immunoassay for C-Reactive Protein, and novel reagents useful in the assay.
2. Background Art
Competitive binding immunoassays for measuring ligands are well known, and are based on the competition between a ligand in a test sample and a labeled reagent, referred to as a tracer, for a limited number of receptor binding sites on antibodies specific to the ligand and tracer. The concentration of ligand in the sample determines the amount of tracer that will specifically bind to an antibody. The amount of tracer-antibody complex produced can be quantitatively measured and is inversely proportional to the quantity of ligand in the test sample.
Fluorescence polarization immunoassay techniques are based on the principle that a fluorescent labeled compound, when excited by plane polarized light, will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Specifically, when a molecule such as a tracer-antibody complex having a fluorescent label is excited with plane polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time when light is absorbed and when it is emitted. When a "free" tracer compound (i.e., unbound to an antibody) is excited by plane polarized light, its rotation is much faster than that of the corresponding tracer-antibody complex; therefore, the emitted light is depolarized to a much greater extent. Thus, fluorescence polarization provides a quantitative means for measuring the amount of tracer-antibody complex produced in a competitive binding immunoassay.
Fluorescence polarization techniques have been applied to U.S. Patent No. 4,420,568 to Wang, et al., commonly assigned herewith, which is directed to the use of a triazinylamino-fluorescein moieties as fluorophores. Various other fluorescent labeled compounds are known in the art. For example, U.S. Patent No. 3,998,943 describes the preparation of a fluorescently labeled insulin derivative using fluorescein isothiocyanate (FITC) as the fluorescent label and a fluorescently-labeled morphine derivative using 4-aminofluorescein hydrochloride as the fluorescent label. Carboxyfluorescein has also been used for analytical determinations. R. C. Chen, Analytical Letters, 10, 787 (1977) describes the use of carboxyfluorescein to indicate the activity of phospholipase. The carboxyfluorescein is described as encapsulated in lecithin liposomes, and as fluorescing only when released by the hydrolysis of lecithin. U.S. Patent No. 4,476,229 to Fino, et al., also commonly assigned herewith, describes a series of amino acid amido derivatives of carboxyfluorescein useful as reagents in fluorescence polarization immunoassays.
As previously explained, fluorescence polarization techniques are based on the principle that a fluorescent labeled compound in solution, when excited by plane polarized light, will emit fluorescence having a degree of polarization related to its molecular rotational relaxation time. The molecular rotational relaxation time, and hence the magnitude of the fluorescence polarization response, is directly related to the molecular size of the compound. Accordingly, when plane polarized light is passed through a solution containing a relatively high molecular weight fluorescent compound, the degree of polarization of the emitted light will in general be greater than when plane polarized light is passed through a solution containing a low molecular weight fluorescent compound.
The fluorescence polarization principle is ordinarily utilized in an assay by mixing a sample containing an analyte (or suspected of containing an analyte) with a "tracer,", i.e., a labelled compound similar to the analyte but capable of producing a fluorescence polarization response to plane polarized light. Conventionally, the analyte is a relatively low molecular weight compound, i.e. less than about 2,000 daltons. Antibody specific to the analyte and the tracer is also included in the mixture. The tracer and the analyte compete for a limited number of receptor binding sites on the antibody. The amount of tracer that will bind is inversely related to the concentration of analyte in the sample, because the analyte and tracer each bind to the antibody in proportion to their respective concentrations.
The principle of fluorescence polarization has been successfully applied because it is known that the fluorescence polarization response of an assay solution to plane polarized light will give a quantitative indication of the relative amount of free and bound tracer, because of the discrepancy in molecular size between the former and the latter. The free tracer (i.e., the tracer in solution when not complexed to the antibody) is generally a relatively small molecule compared to the tracer-antibody complex, and thus will tend to exhibit a shorter rotational relaxation time, such that the incident plane polarized light becomes depolarized. In contrast, plane polarized light interacting with bound tracer will tend to remain highly polarized because the large antibody-tracer rotates very little between the time that light is absorbed and emitted.
However, fluorescence polarization techniques have been only applied with reasonable success to the measurement of analytes of relatively low molecular weight. Since the tracer employed must generally resemble the analyte in order to compete effectively for antibody receptor sites, the tracer itself, in such instances, will be relatively large and will tend to retain the polarization of plane polarized light. This approach of doing a competitive binding immunoassay using a fluorescein-labeled compound as the tracer generally works well because of the substantial difference in polarization observed when the tracer is free versus when it is bound by specific antibody. A polarization unit change from 0.02 (free) 0.20 (bound) can be considered typical of many sun tracers.
When a large tracer molecule is bound to an antibody, there will generally not be an appreciable difference in the fluorescence polarization response when compared with the response produced by the free tracer, so that in cases where it has heretofore been desired to measure relatively large molecular weight analytes, i.e., on the order of greater than about 100,000 daltons, it has been generally necessary to consider alterative assay techniques, such as nephelometry. A few examples of fluorescence polarization immunoassays applied to quantitation of higher molecular weight (treater than about 10,000 daltons) substances can be found. Some literature demonstration such assays are H. Maeda, Clin. Chem. 24:2139 (1978); W. B. Dandliker, et al., Immunochemistry 10:219 (1973); and S. A. Levison, et. al., Endocrinology 99:1129 (1976). However, notable features of these disclosures of polarization assays of relatively low molecular weight proteins (less than 100,000 daltons) are the small polarization changes observed when the tracer if free versus when it is bound by antibody. Typically the polarization changes observed a only in the range of 0.015 to 0.045 polarization units.
In contrast, the CRP/fluorescein conjugate tracer described in this disclosure (M.W. 120,000 daltons) exhibits a polarization change of slightly more than 0.10 polarization units.
Nephelometric techniques have been found to provide a satisfactory means for measuring the light scattered from a solution containing large molecules or suspended particles. In accordance with these techniques, incident light is passed through a solution, a portion of the incident light is scattered, and then the amount of scattered light is measured. These techniques have application, for example, when immunoprecipitation assays are conducted. In such assays, antibodies are raised to the analyte, often forming large three-dimensional lattices. These lattices produce an increase in the light scattering properties of the solutions.
The TD.times.@ Fluorescence Polarization Analyzer, an instrument commercially available from Abbott Laboratories, Abbott Park, Illinois, for example, provides the capability of automation of fluorescence polarization assays, and, with minor modification of the instrument, of nephelometric analysis, as well as other systems of analysis. However, it would be desirable and useful to be able to perform fluorescence polarization techniques to measure large molecules, so that, among other reasons, the modifications which are necessarily made to such analyzers to enable them to perform nephelometric as well as fluorescence polarization assays would be unnecessary; assays of large molecular weight species could then be performed using such readily available, unmodified analytical instrumentation.
C-reactive protein (CRP) is a large molecular weight species which it would be advantageous to measure by fluorescence polarization techniques, so that nephelometric or other more complex assay systems would be unnecessary. CRP is a plasma protein synthesized by hepatocytes and is present in the serum of healthy subjects in trace amounts. It is acute-phase protein; within hours of an acute injury or the onset of most types of infection or inflammation, its rate of synthesis and secretion in subjects increases markedly, with a resultant rise in the serum concentration. The amount of the rise has been found to correlate well with the severity of tissue damage.
Precise measurement of serum CRP concentration provides the clinician with a sensitive indication of many diseases. This is of value both in the initial evaluation of a disorder and in monitoring its response to therapy. However, CRP assays fall into a different category from the vast majority of in vitro diagnostic tests, since CRP concentration provides a screening test for organic disease and an indication of disease activity when the diagnosis is known, rather than a indication of a given disease state.
The CRP molecular (molecular weight 120,000) consists of five identical nonglycosylated polypeptide subunits. The capacity of CRP to bind a wide range of different ligands is presumably central to its in vivo function. Thus CRP is conventionally detected and quantitated by various immunological methods. Tests based on the interaction between CRP and pneumococcal C polysaccharide, and capillary tube precipitation methods involving anti-CRP serum, are well known. The simpliest current method is a latex-agglutination procedure, in which latex particles coated with anti-CRP antibodies are agglutinated by CRP in the serum. Although quite sensitive (detection limit about 5 mg/l) and rapid, the latex test is only qualitative, and is subject to technical problems and interferences. Accordingly, it does not provide as valuable information as that furnished by precise, quantitative methods.
The classic immunochemical techniques of radial immunodiffusion and electroimmunoassay can be used to measure CRP accurately and with suitable sensitivity, but they are slow and technically demanding for routine work. Ideally, a CRP assay should be rapid and technically simple, yield a accurate result, and be capable of automation for large-scale use. A number of such systems are commercially available based on either homogeneous enzyme, fluoroimmunoassay or rate immunephelometry. However, heretofore the economy, ease of performance and simplicity of quantitation of CRP by fluorescence polarization immunoassay has not been known, because of the aforedescribed constraints placed upon such techniques by the relatively large size of the CRP molecule.